Pyruvate (Pyr) is a normal component in the body of human, animals and plants. It is a product of glycolysis, which is a biological basic metabolism. It is at the convergence of metabolisms of sugar, fat and protein and it is a critical starting substance for Tricarboxylic Acid Cycle (TCA cycle). In recent decades, large numbers of in vitro and in vivo animal experiments and a few clinical trials strongly showed that sodium pyruvate (Sod Pyruvate, NaPyr) can substantially protect functions of multiple cell lines and organs, particularly ischemia-reperfusion injury of some important organs, such as heart, liver, brain and kidney, without toxic side effects (Am J Physical 281:H854-64, 2001; Lancet 353:1321-3, 1999; Neurobiol Dis 26:94-104, 2007).
Otherwise, recent studies suggested that NaPyr would be a superior buffer for correcting severe metabolic acidosis. The severe metabolic acidosis is severe clinical conditions, such as type A (anoxic type) lactic acidosis, diabetic ketoacidosis and hypercapnia with cardiac arrest, which comprise more than 90% of severe metabolic acidosis (pHa ≦7.10). Heretofore, no ideal buffer has been used in the art worldwide. Numerous in vitro and in vivo animal experiments demonstrated that NaPyr has special effects on improving intracellular and extracellular acidosis. Noticeably, NaPyr could exert important effects even under anoxic conditions, which is particularly suitable for the above mentioned severe acidosis. More importantly, NaPyr is not only a buffer, it also has direct therapeutic effects on dysfunction of primary affected organs causing the above mentioned severe conditions. It makes NaPyr completely different from sodium lactate, which has been used for more than half a century, and sodium bicarbonate, which has been used longer. Both sodium lactate and sodium bicarbonate are not suitable for the above mentioned severe acidosis, and they have toxicity (Am J Nephrol 25:55-63, 2005; Am J Kidney Dis 38:703-27, 2001).
Clinically, Pyr seems to be a potential effective medicine, especially in the field of intensive care and dialysis. However, an extensively accepted conception is that aqueous solution containing pyruvate or sodium pyruvate is very unstable at room temperature, because the chemical property of keto is unstable, which can produce dimer (4-hydroxy-4-methyl-2-ketoglutaric acid, also known as parapyruvate) and other polymeric derivatives by spontaneous aldol-like condensation and a small amount of hydrates by hydration in non-enzyme catalysis. The polymer is cytotoxic, and is an inhibitor of the enzyme in TCA cycle in mitochondria and lactate dehydrogenase (LDH). The hydrate makes Pyr lose its metabolic activity. The conclusion made 20 years ago by the authoritative report of NBS: Center for Analysis Chemistry, National Bureau of Standards, USA was that aqueous solution containing NaPyr is unstable at 22° C., pH >6.0, as determined by High Performance Liquid Chromatography (HPLC) and Nuclear Magnetic Resonance (NMR). At room temperature and pH 7.3, the content of dimer can reach 9.1% in high concentration 3.0 M (3,000 mM) of pure NaPyr in water after 24 hours, and can reach 18.4% after 48 hours (Anal Chem 58:2504-10, 1986). It is commonly considered that the perfusion solution containing NaPyr should be prepared freshly and be used as soon as possible. It was indicated in US patent 20050058724 that the aqueous solution containing pyruvate anion could not be used as therapeutic fluids for human. Some studies indicated that under low temperature (4-8° C.), 0.1M HCl or 0.1 M NaCl could make aqueous solution containing NaPyr stable. The effect of various concentrations of HCl or NaCl on the stability of NaPyr under room temperature or high temperature (e.g. 40° C.) was not shown. Room temperature or high temperature is a common condition for clinical usage, manufacturing or storage. Therefore, no pharmaceutical company could produce aqueous solution containing stable Pyr at room temperature in the world, including intravenous injection solution and dialysis solution. Except for some clinical trials, aqueous solution containing NaPyr has never been used in routine clinical practice.
In order to solve the above problem and deliver a large amount of Pyr into the body to achieve the aim of treatment, the direction of R&D in recent years (i.e. since the brief report originally published on Surgical Forum in 1999) is to develop ester derivatives of Pyr, such as Ethyl Pyruvate (EP), which is Ringer's solution with NaLac replaced by EP (Ringer's Ethyl Pyruvate Solution, REPS) and Dipyruvyl Acetyl Glycerol (DPAG). Some in vivo experiments showed their inspiring therapeutic effects (Minerva Anestesiol 67:190-2, 2001, Cardiovasc Drugs Ther 17:209-16, 2003), and they were also filed for patent protection, as described in U.S. Pat. No. 6,846,842. However, the developments in these aspects also hold back further investigation of the stability of aqueous solution containing NaPyr. The ester derivatives of Pyr still have the following disadvantages although the research of EP is in full swing: 1, EP can be solved and stabilized only by binding with calcium ion, and it should be solved in Ringer's equilibrium solution which contains calcium chloride (2.7 mEq/L) to be used in laboratories (Crit Care Med 29:1513-8, 2001), and its concentration is only 28 mM and could not be prepared as isotonic and hypertonic solution purely containing EP. 2, Ester cannot be ionized as Pyr− in water, but can be spontaneously hydrolyze as Pyr− through esterase or non-enzyme pathway (thereby producing hydrogen ion [H+]). In contrast to NaPyr, esters of Pyr are not buffer and can produce the same amount of [H+] through in vivo hydrolysis. In vivo tests of shocked animal showed that although shock was improved with REPS, EP, per se, could not correct acidosis (Shock 18:507-12, 2002) and EP could not be prepared as a buffer used in dialysis solution, either. 3, The concentration of chloride ion in REPS was still so high that transfusion of a large volume of REPS would lead to hyperchloremic acidosis. 4, On the market, as a raw material, the esters of Pyr are not as plentiful as sodium salt of Pyr. Thus, the esters of Pyr cannot replace aqueous solution containing NaPyr mostly in clinical practice.
Therefore, there is an urgent need in the art to provide a stable aqueous solution containing Pyr, which can deliver the Pyr with superior features into the body effectively, i.e. improve metabolic pathways in vivo, together with protecting vital organs' functions and improving metabolic disorders.